Team:Paris Saclay/Notebook/August/28

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Latest revision as of 01:20, 5 October 2013

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Contents

Notebook : August 28

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000 and BBa_K1155004

1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in pSB1C3, NirB with RBS-Amil CP-Term in pSB1C3, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions

XiaoJing

Transformation of 08/26/13 works. We will use it to characterize all ligations. PCR colony of 08/27/13 works for . We also will use them to characterize ligations : NirB with RBS-Amil CP-Term in pSB1C3, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3.

We streak colonies from construction :

  • Pndh* with RBS-LacZ-Term in pSB1C3 with O2 and Xgal
  • Pndh* with RBS-Amil CP-Term in pSB1C3 with O2
  • Pndh* with RBS-Amil CP-Term in pSB1C3 without O2
  • NirB with RBS-LacZ-Term in pSB1C3 without O2 and Xgal
  • NirB with RBS-Amil CP-Term in pSB1C3 with O2
  • NirB with RBS-Amil CP-Term in pSB1C3 without O2

2 - Culture of mutant strain MG1655Z1 Δfnr

XiaoJing

Transformation of 08/27/13 works. We will do a purification on LB plates.

We streaked our colonies on plates with LB and incubated them at 42°C.

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FRN and BphR2 proteins

1 - Gel purification of pSB1C3 digested by DnpI

XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • pSB1C3 : 37.2ng/µL

2 - Electrophoresis to check the gel purification of pSB1C3 digested by DnpI

XiaoJing

Psgel12808.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of pSB1C3 digested by DpnI + 1µl of 6X loading dye
  • Gel : 1%

expected sizes :

  • pSB1C3 : 2070 bp

We obtained a fragment at the right size. The gel purification was good. We will use it for Gibson assembly.

3 - Gibson assembly

XiaoJing

Used quantities :

  • RBS-BphR2 :
    • pSB1C3 : 3µL
    • BphR2 Part I : 1µL
    • BphR2 Part II : 1µL
    • Gibson mix : 15µL
  • FNR :
    • pSB1C3 : 3µL
    • FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gisbon mix : 15µL
  • RBS-FNR :
    • pSB1C3 : 3µL
    • RBS-FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gibson mix : 15µL

We incubate these mix at 50°C during 1h inside PCR machine.

2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α strain

XiaoJing

Protocol : Bacterial transformation


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